Within the scope of the present invention, the term microfluidic channel refers to a closed channel, i.e. an elongated passage for fluids, with a cross-section microscopic in size, i.e. with the largest dimension of the cross-section being typically from about 1 to about 500 micrometers, preferably about 10 to about 300 micrometers. A microfluidic channel has a longitudinal direction, that is not necessarily a straight line, and that corresponds to the direction in which fluids are flowing within the microfluidic channel, i.e. preferably essentially to the direction corresponding to the average speed vector of the fluid, assuming a laminar flow regime.
A microcarrier or a microparticle refers to any type of particles, respectively to any type of carriers, microscopic in size, typically with the largest dimension being from 100 nm to 300 μm, preferably from 1 μm to 200 μm.
According to the present invention, the term microcarrier refers to a microparticle functionalized, or adapted to be functionalized, that is containing, or adapted to contain, one or more ligands or functional units bound to the surface of the microcarrier or impregnated in its bulk. A large spectrum of chemical and biological molecules may be attached as ligands to a microcarrier. A microcarrier can have multiple functions and/or ligands. As used herein, the term functional unit is meant to define any species that modifies, attaches to, appends from, coats or is covalently or non-covalently bound to the surface of said microcarrier or impregnated in its bulk. These functions include all functions that are routinely used in high-throughput screening technology and diagnostics.
Drug discovery or screening and DNA sequencing commonly involve performing assays on very large numbers of compounds or molecules. These assays typically include, for instance, screening chemical libraries for compounds of interest or particular target molecules, or testing for chemical and biological interactions of interest between molecules. Those assays often require carrying out thousands of individual chemical and/or biological reactions.
Numerous practical problems arise from the handling of such a large number of individual reactions. The most significant problem is probably the necessity to label and track each individual reaction.
One conventional method of tracking the identity of the reactions is achieved by physically separating each reaction in a microtiter plate (microarray). The use of microtiter plates, however, carries several disadvantages like, in particular, a physical limitation to the size of microtiter plates used, and thus to the number of different reactions that may be carried out on the plates.
In light of the limitations in the use of microarrays, they are nowadays advantageously replaced by functionalized encoded microparticles to perform chemical and/or biological assays. Each functionalized encoded microparticle is provided with a code that uniquely identifies the particular ligand(s) bound to its surface. The use of such functionalized encoded microparticles allows for random processing, which means that thousands of uniquely functionalized encoded microparticles may all be mixed and subjected to an assay simultaneously. Examples of functionalized encoded microparticles are described in the international patent application WO 00/63695 and are illustrated in FIG. 1.
The international patent application WO 2010/072011 describes an assay device having at least one microfluidic channel which serves as a reaction chamber in which a plurality of functionalized encoded microparticles or microcarriers can be packed. Typically, such a microcarrier 1, illustrated in FIG. 1, comprises a body 2 having a shape of a right circular cylinder or disc delineated by a first circular surface 3 and a second circular surface, not shown, opposite to the first circular surface 3. Such a microcarrier 1 is usually encoded by a distinctive mark attached to it for its identification. The distinctive mark may comprise a distinctive pattern of a plurality of traversing holes 4 and may also include an asymmetric orientation mark 5 such as, for example, a L-shaped sign or a triangle, as shown in FIG. 1. This asymmetric orientation mark 5 allows the distinction between the first circular major surface 3 and the second circular major surface.
The microfluidic channel of the assay device described in WO 2010/072011 is provided with stopping means acting as filters that allow a liquid solution containing chemical and/or biological reagents to flow through while blocking the microcarriers 1 inside. The geometrical height of said microfluidic channel and the dimensions of said microcarriers are chosen so that said microcarriers 1 are typically arranged in a monolayer arrangement inside each microfluidic channel preventing said microcarriers 1 to overlap each other.
The European patent application EP11000970.1 describes an encoded microcarrier 6 as shown in FIG. 2, the first circular surface 3 of said microcarrier 6 comprising a detection surface 8 to detect a chemical and/or biological reaction and further comprising protruding means 7 which are shaped to ensure that, when the encoded microcarrier 6 is laid on a flat plane with the detection surface 8 facing said flat plane, a gap exists between said flat plane and this detection surface.
The detection of a reaction of interest can be based on continuous readout of the fluorescence intensity of each encoded microcarrier present in a microfluidic channel of an assay device. The presence of a target molecule in the assay will trigger a predetermined fluorescent signal which is detected through a transparent observation wall of the assay device. When an encoded microcarrier is injected in the microfluidic channel, its detection surface is intended to face said observation wall and a laminar flow of liquid (containing chemical and/or biological reagent of interest for the assay) is intended to pass through the above-mentioned gap between said detection surface and the observation wall. Thanks to this laminar flow of liquid in the gap, the microcarrier presents a more homogeneous reaction of interest on its detection surface.
As shown in FIG. 3, the microcarriers 6 are prepared in suspension in a liquid sample 16 which is injected in a microfluidic channel 13 via an inlet well 14 having a sidewall 15 on which opens out an end of the microfluidic channel 13. The bottom wall 17 of the inlet well 14 is connected to a microfluidic channel bottom wall 18 which comprises the above-mentioned observation wall 10.
In the prior art, the liquid sample 16 is injected in the microfluidic channel 13 by injecting means which has a tip 19 through which the liquid sample is intended to exit when being injected, said tip 19 being inserted into the inlet well 14 during injection. During said injection, the liquid sample 16 comes into contact with the bottom wall 17 of the inlet well 14, and the microcarriers 6 deposit by sedimentation from the tip 19 until they land on the bottom wall 17 of the inlet well 14. The detection of the presence of molecules bound to the detection surfaces 8 is only possible when said detection surfaces 8 face the observation wall 10, as shown by a first microcarrier 11 in the FIG. 4. However, during sedimentation, the microcarriers 6 may flip over so that some of the microcarriers 6 present their detection surface 8 opposite to the observation wall 10 of the microfluidic channel 13, as a second microcarrier 12 shown in FIG. 4. Thus, the second microcarrier 12 presenting a wrong orientation of its detection surface cannot emit any detectable signal and can be considered as false negative during the biological assay. Moreover, the fluid flow, represented by the arrows B is disturbed by the second microcarrier 12, which does not present a spacing 9 between its detection surface 8 and the observation wall 10. Indeed, in the absence of the spacing 9, the velocity of the fluid flow is very low in the vicinity of the wall 10. The velocity field of the fluid flow is then inhomogeneous in the microfluidic channel 13 which led to an inhomogeneous distribution of the reagents and target molecules intended to interact with the detection surfaces 8 of the first microcarrier 11 (since the reagents are not renewed in the fluid flow portions where the velocity is very low). Thus, it is of major importance to prevent the problem of the wrong orientation of the microcarriers within the microfluidic channel for performing a reliable biological assay for research and clinical laboratories.